We have discovered that the intermediate carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) is converted to a 2-nitrosofluorene (NOF)-lipid adduct (N-O-LAF) in rat liver microsomes and nuclei. N-O-LAF is also formed from the proximate carcinogen N-acetoxy-2-acetylaminofluorene. It appears that deacylase enzymes control the rate of N-O-LAF formation from these carcinogens and that this process is considerably decreased by the antioxidants, butylated hydroxytoluene and vitamin E as well as p-hydroxy-acetanilide. Two of these compounds have been shown to decrease tumor formation by these carcinogens. We will investigate the molecular events involved in N-O-LAF formation and determine which lipid component in the natural membrane the carcinogen becomes bound to and if this carcinogen-lipid adduct is mutagenic in the Ames mutagenesis assay. We propose to determine if NOF adds to DNA in an "ene" type reaction similar to what we have discovered for NOF addition to double bonds of lipid compounds. We also want to determine if NOF adds to free radicals of DNA in a spin-trapping type reaction. We have discovered that autooxidizing ascorbate converts NOF into non-mutagenic products. We will investigate the chemical events involved. They appear to involve a spin-trapping reaction i.e., the addition of a free radical to a c-nitroso compounds. We propose to elucidate the events involved in a discovery we have made, namely the demonstration that rat liver nuclei activate N-OH-AAF into mutagenic products. This will be investigated in detail.